Department of Natural Resources and Environmental Sciences, University of Illinois, Champaign, Illinois 61820, USA DAVID P.

Transcription

1 Joural of Aquatic Aimal Health 17: , 2005 Copyright by the America Fisheries Society 2005 DOI: /H [Article] Effects of Factors Related to Water Quality ad Populatio Desity o the Sesitivity of Juveile Largemouth Bass to Mortality Iduced by Viral Ifectio KATE R. INENDINO AND EMILY C. GRANT Departmet of Natural Resources ad Evirometal Scieces, Uiversity of Illiois, Champaig, Illiois 61820, USA DAVID P. PHILIPP Ceter for Aquatic Ecology, Illiois Natural History Survey, ad Program i Ecology ad Evolutioary Biology, Uiversity of Illiois, Champaig, Illiois 61820, USA TONY L. GOLDBERG* Departmet of Veteriary Pathobiology, College of Veteriary Medicie, ad Program i Ecology ad Evolutioary Biology, Uiversity of Illiois, Champaig, Illiois 61820, USA Abstract. Evirometal stressors ca predispose fish to mortality from ifectious disease. This study examied the effects of two factors, water quality ad physical crowdig, o the resposes of fish to viral ifectio. Juveile largemouth bass Micropterus salmoides were experimetally ioculated with largemouth bass virus (LMBV), a emergig pathoge i the family Iridoviridae. I separate experimets, fish were exposed to various cocetratios of itrate (0, 40, 200, ad 400 mg/l) ad were housed at either high or low populatio desities. Survival time, viral load (quatity of virus i tissues), ad body coditio were measured as outcomes. Nitrate, as well as other water quality parameters such as ammoia, itrite, ad ph, affected mortality rates ad viral loads i complex ways. Paradoxically, icreased itrate cocetratios were associated with reduced mortality rates i juveiles exposed to LMBV. I geeral, rapid fluctuatios i the cocetratios of dissolved toxis had greater impact o sesitivity to viral ifectio tha did persistetly high levels of these toxis. Fish housed at high desity experieced icreased mortality rates, elevated viral loads, ad reduced body coditio compared with fish held at low desity. These results demostrate that both physiochemical ad social evirometal stressors ca affect the survival ad coditio of largemouth bass ifected with LMBV. Adverse evirometal coditios ca affect the health ad growth of fish both i captivity ad i the wild. Suboptimal water quality ad high populatio desities, i particular, are kow to affect fish health egatively (Nicholso et al. 1990; Carballo ad Muoz 1991; Wedemeyer 1996). Reduced water quality exacts a direct physiological toll o fish. Both cocetratios of oxious chemicals ad how log the fish are exposed to them determie the extet of physiological damage (Carballo ad Muoz 1991). Fish livig i crowded coditios ca become stressed from either altered social iteractios or restrictios of their ability to move freely or to otherwise behave ormally (Wedemeyer 1996). * Correspodig author: tlgoldbe@uiuc.edu Received May 13, 2004; accepted March 4, 2005 Published olie August 24, 2005 Water quality reductios ad demographic factors may also affect the ability of fish to resist ifectious disease. Sublethal cocetratios of toxis or pollutats are associated with immuosuppressio ad susceptibility to ifectious disease i fish (e.g., Hetrick et al. 1979; Kittel 1981; Carballo et al. 1995; Arkoosh et al. 1998). Nitrate, i particular, may suppress fish immuity either directly, through egative effects o fuctioal immuity, or idirectly, through geeral physiological stress (Hrubec et al. 1996). Fish held i high desities are kow to suffer immuosuppressio (Meade 1986; Pickerig ad Pottiger 1987). High fish desity has bee associated with disease epidemics i coho salmo Ocorhychus kisutch (Fagerlud et al. 1986), Chiook salmo O. tshawytscha (Mazur et al. 1993; Baks 1994), white sturgeo Acipeser trasmotaus (LaPatra et al. 1996), ad raibow trout O. mykiss (Bebak- Williams et al. 2002). Nevertheless, the mecha- 304

2 VIRAL INFECTION OF LARGEMOUTH BASS 305 isms by which water-quality reductios ad demographic factors reduce resistace to ifectious disease are poorly uderstood. Viruses of the family Iridoviridae are of icreasig cocer for both wild ad farmed fish (Williams 1996; Georgiadis 2001; Chichar 2002). Iridoviruses are large, complex DNA viruses that cause systemic ifectios ad mortality i fish (Wolf 1988), amphibias (Daszak et al. 1999; Docherty et al. 2003), reptiles, ad isects (Williams 1996; Chichar 2002). Large-scale mortality evets attributable to iridoviral disease have occurred i wild populatios of amphibias ad fish (Daszak et al. 1999). Iridovirus epidemics have also caused ecoomic losses i commercial aquaculture systems (Lagdo ad Humphrey 1987; Williams 1996; Essbauer ad Ahe 2001; Georgiadis 2001). Iridoviral ifectio causes extremely varied cliical resposes i fish, ragig from lethal to subcliical. The causes of this variability are poorly uderstood, although evirometal factors are suspected to be critical determiats of cliical disease i fish ifected with iridoviruses (LaPatra et al. 1996; Chichar 2002; Goldberg 2002). This study addresses the hypotheses that reduced water quality ad crowded coditios ca alter the sesitivity of fish to mortality from iridoviral ifectio ad that such effects operate through immuosuppressive mechaisms to facilitate viral replicatio withi fish. We performed two separate viral challege experimets to test this hypothesis. The first experimet ivestigated the effects of selected water quality parameters o the severity of cliical disease ad viral loads i experimetally ifected fish. The secod experimet ivestigated the effects of high populatio desity o the severity of cliical disease ad viral loads i similarly ifected fish. We used as a study pathoge largemouth bass virus (LMBV), a emergig iridovirus that has caused mass mortality evets i largemouth bass Micropterus salmoides ad Florida bass M. floridaus throughout the souther ad easter Uited States (Goldberg 2002; Grizzle ad Bruer 2003). Like other iridoviruses, LMBV varies widely i the ature ad severity of cliical disease it causes (Haso et al. 2001; Goldberg 2002). We predicted that reduced water quality ad icreased populatio desity would both adversely affect the ability of ifected fish to cotrol viral replicatio ad to resist cliical disease. Methods Study orgaisms. The juveile largemouth bass used i all experimets came from experimetal rearig pods at the Illiois Natural History Survey s Aquatic Research Field Laboratory, which is located o the Uiversity of Illiois Urbaa-Champaig campus. All fish were betwee 4 ad 6 moths of age with a mea total legth of mm ( SE) ad a mea weight of g. Fish were origially collected from the Kaskaskia River basi i Illiois. This stock has bee reared for several geeratios at the pod site ad has ot bee exposed to LMBV. Before the study, we tested a represetative sample of fish from this populatio for LMBV, usig virus isolatio. All fish tested were egative. Experimetal fish were held i either 10-gal (37.8-L; water quality experimet) or 20-gal (75.6-L; desity experimet) aquaria housed iside a cotrolled eviromet chamber kept at costat temperature (25 C) ad photoperiod (12 h light : 12 h dark). Fish were fed bloodworms (approximately 0.8 g/ fish) daily at 0900 hours, except o the day of exposure to LMBV. Virus. The viral isolate used for ioculatio of largemouth bass was a sixth cellculture passage of the origial type isolate recovered from the 1995 LMBV Satee Cooper Reservoir fish kill i South Carolia (Plumb et al. 1996). Virus was grow ad harvested accordig to previously published methods (Goldberg et al. 2003). Experimetal ioculatio. Experimetal fish received 0.1 ml of Hak s Balaced Salt Solutio (HBSS) cotaiig live virus at a cocetratio of geomes of LMBV per dose, measured by usig real-time quatitative polymerase chai reactio (PCR). This is the dose calculated to kill half the experimetal subjects (LD50) withi 2 weeks postexposure (Goldberg et al. 2003). Sham-ijected cotrol fish received the same volume (0.1 ml) of udiluted virus-free cell culture superatat. Fish received ijectios i the peritoeal cavity, alog the vetral midlie halfway betwee the pelvic ad aal fis. Moitorig ad processig fish. We moitored survival of the fish for 2 weeks after ijectios. At 24-h itervals, we recorded the umber of dead fish i each tak ad recorded qualitative data o the behavior ad appearace of survivig fish. We fed fish daily with approximately 0.2 g of pure froze bloodworms (red mosquito larvae; Sa Fracisco Bay Brad, Newark, Califoria) per fish. We removed dead ad moribud fish daily from

3 306 INENDINO ET AL. the taks. We killed moribud fish (fish that were listless or swimmig with marked loss of equilibrium) i a solutio of clove oil emulsified i ethaol (1:9 by volume; Taylor ad Roberts 1999). We recorded the total weight (g) ad total legth (mm) of each fish, as well as the presece of ay exteral or iteral gross pathological lesios. We quatified the body coditio of each fish at the time of death accordig to Fulto s idex [K weight i g (total legth i mm) 3 100,000 1 ], a commo measure for comparig the coditio of fish withi populatios (Bolger ad Coolly 1989). We the removed the viscera (liver, splee, stomach, itesties, kidey, swim bladder, heart, ad reproductive orgas) ad diluted them 1:50 i HBSS cotaiig atibiotics (peicilli, 100 uits/ ml; getamyci sulfate, 50 g/ml; streptomyci, 100 g/l; ad amphoterici B, 0.25 g/l). We homogeized the viscera with a stomacher (Stomacher 80 Biomaster automatic stomacher; Steward, Ltd., Worthigto, UK) at ormal speed for 1 mi ad froze the tissue homogeates at 80 C. We quatified viral load by usig real-time quatitative PCR, expressig the results as the umber of viral geomes per gram of fish tissue, accordig to published protocols (Goldberg et al. 2003). Water quality experimet. We performed a prelimiary experimet to determie what cocetratio of itrate was toxic to the fish used i this study. We placed 10 juveile largemouth bass each ito four 10-gal (37.8-L) aquaria equipped with recirculatig water supplies ad air stoes ad held them at 25 C. We the added sodium itrate solutio (1 M) to each tak, at differet rates (0, 10, 40, ad 100 mg/l per day). The fial cocetratios of sodium itrate added to these taks raged from 0 to 1,600 mg/l. We observed fish for toxic effects ad termiated the experimet whe fish displayed overt sigs of itrate toxicosis (disequilibrium, spiral swimmig). Fish used i this experimet were ot ioculated with LMBV. We the coducted a experimet to determie the effects of selected water quality parameters (elevated levels of dissolved itrate i particular) o the sesitivity of bass to mortality from LMBV ifectio. For this experimet, we used twelve 20- gal (75.6-L) aquaria, each holdig 10 fish. We kept the aquaria, which were bare except for a aerated filter spoge, iside a cotrolled eviromet chamber at 25 C ad o a timed 12-h-light : 12-hdark photic period. We allowed fish to acclimate to these coditios for 48 h before ay maipulatio. We radomly selected 8 of the 12 taks to serve as experimetal taks (LMBV-ijected fish). The remaiig four taks served as cotrols (shamijected fish). We added itrate to taks to the followig four fial cocetratios of NO 3 -N: 0, 40, 200, ad 400 mg/l. We chose this rage of cocetratios based o our prelimiary toxicity trial, selectig a maximum cocetratio well below that which caused direct toxic effects to bass but well above that typically see i fish farms or i the atural eviromet. We gradually itroduced a solutio of 1 M sodium itrate ito the aquaria at various rates so that the taks attaied their particular target cocetratios after 3 d. For each target itrate cocetratio, we used two of the eight experimetal taks ad oe of the four cotrol taks, such that 20 experimetal fish ad 10 cotrol fish were exposed to each itrate cocetratio. We allowed fish to acclimate to their target itrate cocetratio for four additioal days before ijectig them with LMBV. Water chemistry measuremets. We took daily samples (50 ml) of water from each aquarium ad froze them at 20 C i osterile plastic coical cetrifuge tubes with screw caps (Fisherbrad; Fisher Scietific, Pittsburgh, Pesylvaia). We stored samples for o more tha 2 moths before aalysis. For aalysis, we thawed samples i a 25 C water bath ad filtered them to remove debris ad bacteria through a m pore-size syrige filter uit (millex-hv; Millipore, Molsheim, Frace), usig disposable sterile plastic syriges. We determied the ph of each water sample with a ph meter io electrode at 25 C (the water temperature at which the samples were collected). We determied itrate ad itrite cocetratios by io chromatography (Dioex DX-120 Chromatograph, IoPac AS4A-SC Aio Exchage Colum, Dioex Corporatio, Suyvale, Califoria). Whe ecessary, we diluted samples with deioized water to adjust cocetratios to be withi the limits of the machie stadards (NO 3 -N, NO 2 - N 4.0 mg/l). We measured total ammoia itroge cocetratios (TAN; mg/l) by usig methodology based o the Berthelot Reactio, i which additio of sodium itroprusside forms a blue color that ca be measured with a colorimeter at 660 m (Bra Luebbe TRAACS 2000 Aalytical Cosole, Bra Luebbe Idustrial Method No T; G. Bra Luebbe Ic., Delava, Wiscosi). For all measuremets (NO 2 -N, NO 3 -N,

4 VIRAL INFECTION OF LARGEMOUTH BASS 307 TAN), the miimum detectable level was 0.01 mg/l. Effect of itrate o viral growth i vitro. To measure the direct effects of itrate o viral growth, we grew LMBV i cell cultures at a series of itrate cocetratios idetical to those used for the i vivo study. We ioculated the Satee Cooper LMBV isolate at a multiplicity of ifectio of 1.0 oto cofluet moolayers of fathead miow cells i 25 cm 2 flasks ad added sodium itrate to the cell culture superatat. We used three virusioculated replicate flasks ad oe virus-free cotrol flask per itrate treatmet level (NO 3 -N at 0, 40, 200, ad 400 mg/l) ad icubated them at 25 C. We removed 250- L aliquots of cell culture superatat from the flasks at 24-h itervals for 5 d. We measured viral cocetratios i these superatats by real-time quatitative PCR (Goldberg et al. 2003). Populatio desity experimet. To determie the effects of physical crowdig o the sesitivity of bass to mortality from viral ifectio, we used eight 20-gal (75.6-L) aquaria, each cotaiig 12 fish. Fish were housed at either high or low desity (four taks for each treatmet). The low-desity treatmet ivolved placig 12 fish i each aquarium such that they had uecumbered access to the etire water volume. The high-desity treatmet ivolved placig the same umber of fish i aquaria to which a plastic feestrated barrier had bee istalled at oe average fish legth (4.5 cm) from the frot glass. The barriers were made of medium-stregth shatter-resistat acrylic sheets (Lucite-ES) ad were achored i place with 100% silico sealat (GE, Hutersville, North Carolia). Fish were placed i the space betwee the barrier ad the frot aquarium wall. This desig crowded fish i the high-desity treatmet while keepig water volume equal betwee the two treatmets. To cotrol for the presece of the acrylic sheet, we istalled idetical barriers i each of the lowdesity taks but placed them flush with the rear wall of the aquarium such that they did ot restrict the movemet of fish. Three aquaria (oe at each desity) served as experimetal taks (housig virus-ijected fish), ad oe aquarium at each treatmet level served as a paired cotrol (housig sham-ijected fish). We placed all four aquaria i a cotrolled eviromet chamber ad held them at a costat temperature of 25 C. Aquaria were bare except for a aerated filter spoge ad the plastic barriers. Fish were kept o a timed 12-h-light : 12-h-dark photic period. Fish were allowed to acclimate to desity treatmets for 24 h before ijectio with LMBV or sham-ijectio. Statistical aalyses. The outcome variables icluded survival times of idividual fish, viral loads i idividual fish, body coditio scores of idividual fish, gross pathology i idividual fish, ad mortality rates i aquaria both over the etire experimet ad for specific time itervals. For the water quality experimet, predictor variables icluded itrate, itrite, ammoia, ad ph cocetratios for each aquarium o each experimetal day. We also created predictor variables to represet daily chages i each water quality parameter, as well as chages over the 3 ad 5 d precedig a mortality evet. For the desity experimet, the sigle predictor variable was treatmet (low or high desity). We performed all statistical aalyses with the computer program SAS (SAS Istitute 2004). We used survival aalysis (Cox proportioal hazard regressio) to ivestigate associatios betwee mortality rates ad either water quality parameters or desity treatmet. We also used logistic regressio, where appropriate, to ivestigate associatios of death evets with various water quality parameter predictors. We used liear regressio to ivestigate relatioships betwee tak-specific mortality rates (proportio of remaiig fish dyig o ay give day) ad water quality parameters, as well as for aalyses of viral loads ad body coditio. We adopted repeated-measures models throughout our aalyses to accout for multiple observatios from the same taks over time. We evaluated assumptios ad goodess-of-fit for all statistical models ad performed appropriate trasformatios of the data as ecessary to esure that model assumptios were satisfied. Results Prelimiary Nitrate Toxicity Trial Fish i the aquarium to which itrate was added at the fastest rate (100 mg/l per day) displayed obvious behavioral abormalities whe itrate cocetratio reached 1,600 mg/l. The fish lost equilibrium, had icreased respiratory effort, ad swam without directioal cotrol. These effects may have bee a direct respose to itrate toxicity, or they may have resulted from other toxis that covary with itrate (e.g., ammoia, itrite). No adverse effects were observed i the three aquaria cotaiig lower fial cocetratios of added itrate (0, 160, ad 640 mg/l).

5 308 INENDINO ET AL. TABLE 1. Meas (rages) of water quality parameters i experimetal taks subjected to differet itrate treatmets. All values are milligrams per liter except for ph. Nitrate treatmet (mg/l) ph Total ammoia itroge NO 2 -N NO 3 -N ( ) 0.53 ( ) 5.77 ( ) 0.20 ( ) ( ) 0.20 ( ) 6.29 ( ) 43.3 ( ) ( ) 2.37 ( ) 3.70 ( ) 212 ( ) ( ) 2.84 ( ) 3.01 ( ) 385 ( ) Water Quality Experimet Water quality parameters i the aquaria before the additio of itrate were similar to the values i the rearig pods from which the fish were collected (see Table 1, zero itrate treatmet). Water quality parameters varied withi ad amog aquaria over the 2-week observatio period. Meas ad rages of water quality parameters across all aquaria durig the experimet are summarized i Table 1. Fish ijected with LMBV bega dyig o day 3 postioculatio (Figure 1). Cumulative mortality of LMBV-ijected fish at 14 d postioculatio was 90% for all treatmets except the 200 mg/l treatmet, where cumulative mortality was 70%. Media survival times for LMBV-ijected fish i the 0, 40, 200, ad 400 mg/l itrate treatmets were 6, 6, 11, ad 10 d, respectively. No sham-ijected cotrol fish died i ay of the treatmets except i the 400 mg/l treatmet, where three fish were killed before the ed of the experimet because of their cliical sigs suggestive of itrate toxicity (spiral swimmig). Virus-ijected fish died at a sigificatly faster rate tha sham-ijected fish for FIGURE 1. Survival of juveile largemouth bass ijected with largemouth bass virus ad held i taks with differet cocetratios of sodium itrate added (0, 40, 200, or 400 mg/l). Sham-ijected fish were exposed to virus-free cell culture superatat ad served as cotrols. No mortality occurred i cotrol fish held i the 0, 20, or 200 mg/l taks, so their results were collapsed ito a sigle (solid) lie.

6 VIRAL INFECTION OF LARGEMOUTH BASS 309 TABLE 2. Statistically sigificat relatioships betwee water quality parameters ad mortality evets withi taks. Results are from logistic regressio aalyses i which the outcome variable was whether or ot ay mortality was observed i a give tak o a give day. Oly statistically sigificat results are show. Parameter Estimate a SE a Chi-square P-value Mea Odds ratio Lower limit Upper limit NO b ph c TAN d NO e a Estimates ad SEs refer to regressio parameter estimates from the fial multiple logistic regressio model. b Value of NO 3 -N o the day o which mortality was measured. c Value of ph 24 h before the day o which mortality was measured. d Value of the 24-h chage i total ammoia itroge. e Value of the chage i NO 2 -N over the 5 d before the day o which mortality was measured. each correspodig itrate treatmet (Figure 1). Gross lesios see icluded white exudate i the coelomic cavity, whiteig ad thickeig of the swim bladder, ad discoloratio of the liver, but these lesios were ot cosistetly preset i experimetal or cotrol fish. Nitrate cocetratio was egatively associated with mortality for LMBV-ijected fish (hazard ratio 0.998, , P 0.006). LMBVijected fish i the two lower (0 ad 40 mg/l) itrate treatmet groups died at a rate 2.7 times faster did fish i the higher (200 ad 400 mg/l) itrate treatmet groups ( , P ). Mortality rates for fish i the 0 ad 40 mg/l itrate groups were statistically idistiguishable, as were those for fish at the 200 ad 400 mg/l levels. These effects could have resulted from itrite beig preset i higher cocetratios i the taks with low added itrate tha i the high-added itrate taks (Table 1). The mea values (over 2 weeks) of water quality parameters withi taks had o statistically sigificat associatio with mortality rate. However, several statistically sigificat relatioships emerged betwee time-specific mortality evets TABLE 3. Mea SE viral loads (log 10 viral geomes per gram of fish tissue) of LMBV-ijected largemouth bass by itrate treatmet ad outcome. Values with asterisks are sigificatly greater tha the other value i that row. Nitrate treatmet (mg/l) Foud dead or moribud Viral load Survived util ed of experimet Viral load * * * (whether or ot a tak experieced ay mortality o a give day) ad water quality parameters. Table 2 summarizes these results. Fially, the magitude of chage i itrate cocetratio over a 5- d period (positive or egative) was positively associated with the proportio of fish dyig withi those 5d(t 0.057; P 0.03). No other water quality parameters were sigificatly associated with proportios of fish dyig withi taks. Across all treatmets, viral loads of LMBVijected fish that were foud dead ( log viral geomes per gram of fish tissue) did ot differ sigificatly from viral loads of LMBV-ijected fish that were foud moribud ad subsequetly killed ( log viral geomes per gram). Viral loads of dead ad moribud fish together ( log viral geomes per gram), however, were sigificatly higher tha those of omoribud fish that were killed at the ed of the experimet ( log viral geomes per gram). Viral load data are summarized i Table 3. Viral loads were greater i fish that preseted with exteral lesios tha i fish that preseted without exteral lesios (t 2.79; P 0.007). Fish i the two lowest itrate treatmet groups (0 ad 40 mg/l) had higher viral loads tha fish i the two highest itrate treatmet groups (200 ad 400 mg/l; t 2.42; P 0.018). No other associatios betwee viral load ad itrate treatmet groups were sigificat. Table 4 describes statistically sigificat associatios betwee water quality parameters ad viral loads. All fish, regardless of ijectio type or itrate treatmet, had statistically idistiguishable body coditio scores. The mea Fulto scores for the 0, 40, 200, ad 400 mg/l treatmets were 1.11, 1.12, 1.13, ad 1.06, respectively. Body coditio

7 310 INENDINO ET AL. TABLE 4. Statistically sigificat associatios betwee water quality parameters ad viral loads of fish. Results are from liear regressio aalyses i which the outcome variable was the fial viral load of idividual fish. Oly statistically sigificat results are show. Predictor Parameter estimate a SE a t-value P-value Correlatio b ph 24-h chage d chage TAN (3-d chage) NO 2 3 d before d chage d chage NO 3 24-h chage d chage Chage up to death Total c a Estimates ad SEs refer to regressio parameter estimates from the fial multiple liear regressio model. b Squared semipartial correlatio coefficiet, type II, which idicates the proportio of variatio i the fial regressio model uiquely accouted for by each idividual predictor variable. c Rage of itrate cocetratios over the etire 2-week experimet. score did ot vary i associatio with ay measured water-quality parameter. Effect of Nitrate o Viral Growth i Vitro Flasks i each itrate treatmet had statistically idistiguishable fial viral cocetratios (Pvalues ragig from to for comparisos with results for cotrol flasks). Rates of viral replicatio were statistically uaffected by itrate cocetratios. Table 5 summarizes the effects of itrate cocetratio o viral growth i vitro. Populatio Desity Experimet Fish ijected with LMBV ad held at low desity bega to die o day 1 postioculatio. The media survival time of fish held at low desity was 5 d. At the ed of the 2-week observatio TABLE 5. Viral cocetratios (mea SE) i cell culture flasks ioculated with LMBV at differet itrate cocetratios. Values are expressed as log 10 viral geomes per milliliter of cell culture superatat at the ed of the 5-d growth period. No statistically sigificat differeces existed either i fial viral cocetratios or i the rate of viral replicatio amog itrate levels. Nitrate level (mg/l) a Viral load a Number of replicate flasks per itrate level. period, 32 of 36 fish (89%) held at low desity had died or were foud moribud. Fish exhibited behavior typical of LMBV ifectio, icludig disequilibrium ad lethargy (Plumb ad Zilberg 1999). No sham-ijected cotrols i the lowdesity treatmet died over the 14-d observatio period. Fish ijected with LMBV ad held at high desity bega dyig o day 3 postioculatio. Agai, the media survival time of fish held at high desity was 5 d. At the ed of the 2-week observatio period, 35 of 36 fish (97%) held at high populatio desity had died or were foud moribud. Oe sham-ijected fish held at high desity died durig the 2-week observatio period. LMBV-ijected fish held at high desity were at a sigificatly higher risk of dyig tha LMBV-ijected fish held at low desity ( ; hazard ratio 1.6; P 0.029; Figure 2). Gross lesios were similar to those described for the water quality experimet. Viral loads of LMBV-ijected fish foud dead (mea of 9.4 log viral geomes per gram) did ot differ sigificatly from viral loads of moribud virus-ijected fish that were subsequetly killed (mea, 9.4 log viral geomes per gram; F 0.01; P 0.996). However, fish that died durig the course of the experimet (high ad low desity combied) had sigificatly higher viral loads (mea, 9.4 log viral geomes per gram) tha did fish that survived to the ed of the experimet (mea, 7.5 log viral geomes per gram; F 62.41;

8 VIRAL INFECTION OF LARGEMOUTH BASS 311 FIGURE 2. Survival of juveile largemouth bass ijected with largemouth bass virus ad held at high or low populatio desity. Sham-ijected fish were exposed to virus-free cell culture superatat ad served as cotrols. P ). Fish ijected with LMBV ad held at high desity had sigificatly higher viral loads tha LMBV-ijected fish held at low desity (t 2.19; P 0.016). Mea viral loads are summarized by desity treatmet i Table 6. Exteral ad iteral lesios showed o sigificat associatio with viral load. However, body coditio scores of fish held at high desity were sigificatly lower for virus-ijected fish (1.12 TABLE 6. Mea SE viral loads (log 10 viral geomes per gram of fish tissue) of juveile largemouth bass ijected with LMBV ad held at low or high populatio desity (see text) by outcome. The asterisk idicates that the value was sigificatly greater tha the other value i that colum. Desity Foud dead or moribud Viral load Survived util ed of experimet Viral load Low High * ) tha for sham-ijected cotrols ( ; t 3.82; P ; Table 7). This differece did ot occur for fish held at low desity (Table 7). Discussio Although we expected that icreased cocetratios of dissolved itrate would have adverse effects o the survival of LMBV-ioculated largemouth bass, itrate was paradoxically protective TABLE 7. Mea SE Fulto body coditio scores of juveile largemouth bass ijected with LMBV ad held at low or high populatio desity (see text). The asterisk idicates that the value was greater tha the other value i that row. There were o sigificat differeces withi colums. Desity Sham ijected Fulto score Virus ijected Fulto score Low High *

9 312 INENDINO ET AL. i this study. Higher itrate levels were associated with lower mortality ad lower viral loads i fish exposed to LMBV. Examiatio of the effects of itrate o viral growth i vitro demostrated that itrate cocetratio does ot affect viral replicatio directly. The protective effect of itrate i vivo must have occurred, therefore, from the actio of itrate o the fish themselves. We speculate that itrate may alter physiological processes withi the fish, causig a ihibitio of viral replicatio withi host cells (perhaps by geerally retardig itracellular metabolism, o which viral replicatio depeds). I geeral, relatioships betwee water quality parameters, survival, ad viral loads were varied ad icosistet. Associatios betwee viral load ad ph, itrite, ad itrate were most robust. The directio of these relatioships varied, however, such that geeralizatio across parameters was difficult (Tables 2 ad 4). Furthermore, the umber of statistical aalyses coducted makes it likely that some margially sigificat results appeared to be sigificat because of type I error. Nevertheless, the most robust associatios that emerged ivolved chages i water quality parameters over time, rather tha absolute water quality values (Tables 2 ad 4). This observatio suggests that rapid fluctuatios i water quality may have had greater impact o sesitivity to viral ifectio tha persistetly high levels of dissolved toxis. The effect of water quality o a idividual fish is iflueced by its species, age, size, ad previous exposure to the water parameter i questio (Wedemeyer 1996). Water chemistry parameters withi this experimetal system were dramatically higher tha typical levels foud i upolluted atural waters or levels recommeded by aquaculture stadards. Most recommeded maximum or miimum water-quality stadards have bee based o the physiological eeds of salmoids, which are a highly sesitive group of fish (Wedemeyer 1996). Largemouth bass, however, like other cetrarchids or members of the cypriid family, have a greater tolerace to itrate tha salmoids do ad have bee able to adapt to adverse water quality situatios, icludig eutrophic waters (Smith ad Williams 1974; Lewis ad Morris 1986; Hedricks et al. 1995). That the bass stock used i this experimet exhibited high tolerace to itrate (as illustrated by the prelimiary itrate toxicity experimet) is therefore ot surprisig. The results of the populatio desity experimet idicate that icreased fish populatio desity ca affect the survival, viral load, ad body coditio of fish ifected with a iridovirus. Fish ijected with LMBV ad held at high desity were at a 1.6- fold greater risk of dyig tha LMBV-ijected fish held at low desity. At the ed of 2 weeks, total mortality i LMBV-ijected fish held at high desity was 8% higher tha total mortality i LMBVijected fish held at low desity. Although ot dramatic (Figure 2), these treds were statistically sigificat. The rage of desities examied i this study was limited, however, ad the dose of virus was chose to cause relatively rapid mortality. That a effect of crowdig o mortality was evertheless observed suggests that populatio desity could be a biologically sigificat factor for determiig the survival of largemouth bass ifected with a potetially lethal virus. Previous studies have documeted egative effects of high fish desity o disease-related outcomes (Fagerlud et al. 1986; Mazur et al. 1993; Baks 1994; LaPatra et al. 1996; Bebak-Williams et al. 2002), but the mechaisms accoutig for these associatios have remaied uclear. Specifically, past studies have ot cotrolled for water volume, such that it is ukow whether icreased pathoge trasmissio rates, sociobehavioral stress, or combiatios of the two accout for the treds observed. Our experimetal desig forced fish ito high desity without alterig water volume. The critical parameter that varied, therefore, was the physical space i which fish were housed. The social stress that accompaies crowded coditios ca cause subordiate fish to have impaired leukocyte fuctio ad suppressed immuity (Cooper et al. 1989). Suppressed immuity could, i tur, lead to a iability to ihibit viral replicatio. The observatio that fish held at high desity ot oly died more quickly but also had sigificatly higher viral loads tha fish held at low desity supports such a mechaism. We did ot examie the effects of physical cotact o the trasmissio of the virus betwee idividuals i this study. Icreased fish desity could lead to icreased physical cotact ad therefore icreased trasmissio. I a separate study (Grat et al. 2005, this issue), we have documeted that direct cotact does ideed facilitate viral trasmissio i this system. The body coditio scores of virus-ijected fish held at high desity were lower tha those of shamijected fish held at high desity. This differece did ot exist for fish held at low desity. The combiatio of high populatio desity ad the presece of a ifectious aget resulted i reduced weights ad poor body coditio, while high pop-

10 VIRAL INFECTION OF LARGEMOUTH BASS 313 ulatio desity aloe did ot. This observatio supports the idea that elimiatio of ifectious agets from itesive rearig systems could icrease the overall health ad productivity of fish populatios. The combied results of our water quality ad populatio desity studies suggest that both physiological ad sociobehavioral stressors ca modify the cliical effects of viral ifectio i fish. The mechaisms by which these effects operate are still ukow, but the results of this study implicate immuosuppressio, modulated either through direct physiological pathways (as i the case of water quality effects) or through idirect euroedocrie pathways (as i the case of populatio-desity effects). The fact that both physical ad social factors ca alter the cliical maifestatios of viral ifectio uderscores the eed for holistic approaches to the maagemet of fish populatios (both captive ad wild) that harbor ifectious agets. Strategies that maximize the quality of the physical eviromet i which fish live while simultaeously miimizig sociobehavioral stress should most effectively icrease health ad productivity. Ackowledgmets We thak Todd Royer for assistace with equipmet ad Jes Sadberger for techical support with water-quality laboratory aalyses. The Illiois Coucil o Food ad Agriculture Research ad the Coservatio Medicie Ceter of Chicago provided fudig for this research. Refereces Arkoosh, M. R., E. Casillas, E. Clemos, A. N. Kagley, R. Olso, P. Reo, ad J. E. Stei Effect of pollutio o fish diseases: potetial impact o salmoid populatios. Joural of Aquatic Aimal Health 10: Baks, J. L Raceway desity ad water flow as factors affectig sprig Chiook salmo durig rearig ad after release. Aquaculture 119: Bebak-Williams, J., P. E. McAllister, G. Smith, ad R. Bosto Effect of fish desity ad umber of ifectious fish o the survival of raibow trout fry, Ocorhychus mykiss (Walbaum), durig epidemics of ifectious pacreatic ecrosis. Joural of Fish Diseases 25: Bolger, T., ad P. L. Coolly The selectio of suitable idices for the measuremet ad aalysis of fish coditio. Joural of Fish Biology 34: Carballo, M., ad M. J. Muoz Effect of sublethal cocetratios of four chemicals o the susceptibility of juveile raibow trout to saprolegiosis. Applied ad Evirometal Microbiology 57: Carballo, M., M. J. Muoz, M. Cuellar, ad J. V. Tarazoa Effects of waterbore copper, cyaide, ammoia, ad itrate o stress parameters ad chages i susceptibility to saprolegiosis i raibow trout (Ochorhychus mykiss). Applied ad Evirometal Microbiology 61: Chichar, V. G Raaviruses (family Iridoviridae): emergig cold-blooded killers. Archives of Virology 147: Cooper, E. L., G. Peters, I. Ahmed, M. Faisal, ad M. Choeum Aggressio i tilapia affects immuocompetet leucocytes. Aggressive Behavior 15: Daszak, P., L. Berger, A. A. Cuigham, A. D. Hyatt, D. E. Gree, ad R. Speare Emergig ifectious diseases ad amphibia populatio declies. Emergig Ifectious Disease 5: Docherty, D. E., C. U. Meteyer, J. Wag, J. H. Mao, S. T. Case, ad V. G. Chichar Diagostic ad molecular evaluatio of three iridovirus-associated salamader mortality evets. Joural of Wildlife Diseases 39: Essbauer, S., ad W. Ahe Viruses of lower vertebrates. Joural of Veteriary Medicie, Series B 48: Fagerlud, U. H. M., J. R. McBride, H. Dosajh, T. Bilto, R. B. Morley, ad J. Va Tie Desity effects o performace to release of three sizegroups withi populatios of pod-reared juveile coho salmo: smolts released from Quisam Hatchery i Caadia Techical Report of Fisheries ad Aquatic Scieces Georgiadis, M. P Factors ifluecig trasmissio, oset, ad severity of outbreaks due to white sturgeo iridovirus i a commercial hatchery. Aquaculture 194: Goldberg, T. L Largemouth bass virus: a emergig problem for warmwater fisheries? Pages i D. P. Philipp ad M. S. Ridgway, editors. Black bass: ecology, coservatio, ad maagemet. America Fisheries Society, Symposium 31, Bethesda, Marylad. Goldberg, T. L., D. A. Colema, E. C. Grat, K. R. Iedio, ad D. P. Philipp Strai variatio i a emergig iridovirus of warmwater fishes. Joural of Virology 77: Grizzle, J. M., ad C. J. Bruer Review of largemouth bass virus. Fisheries 28(11): Grat, E. C., K. Iedio, W. J. Love, D. P. Philipp, ad T. L. Goldberg Effects of practices related to catch-ad-release aglig o mortality ad viral trasmissio i juveile largemouth bass ifected with largemouth bass virus. Joural of Aquatic Aimal Health 17: Haso, L. A., L. Petrie-Haso, K. O. Meals, V. G. Chichar, ad M. Rudis Persistece of largemouth bass virus ifectio i a orther Mississippi reservoir after a die-off. Joural of Aquatic Aimal Health 13: Hedricks, S. A., M. J. Maceia, ad W. C. Reeves.

11 314 INENDINO ET AL Abiotic ad biotic factors related to black bass fishig quality i Alabama. Lake ad Reservoir Maagemet 11: Hetrick, F. M., M. D. Kittel, ad J. L. Fryer Icreased susceptibility of raibow trout to ifectious hematopoietic ecrosis virus after exposure to copper. Applied Evirometal Microbiology 37: Hrubec, T. C., J. L. Robertso, S. A. Smith, ad M. K. Tiker The effect of temperature ad water quality o atibody respose to Aeromoas salmoicida i sushie bass. Veteriary Immuology ad Immuopathology 50: Kittel, M. D Susceptibility of steelhead trout Salmo gairderi Richardso to redmouth ifectio Yersiia ruckeri followig exposure to copper. Joural of Fish Diseases 4: Lagdo, J. S., ad J. D. Humphrey Epizootic haematopoietic ecrosis, a ew viral disease i redfi perch Perca fluviatilis i Australia. Joural of Fish Disease 10: LaPatra, S. E., J. M. Groff, T. L. Patterso, W. D. Shewmaker, M. Caste, J. Siple, ad A. K. Hauck Prelimiary evidece of sturgeo desity ad other stressors o maifestatio of white sturgeo iridovirus disease. Joural of Applied Aquaculture 6: Lewis, W. M., ad D. P. Morris Toxicity of itrite to fish: a review. Trasactios of the America Fisheries Society 115: Mazur, C. F., D. Tillapaugh, ad G. K. Iwama The effects of feedig level ad rearig desity o the prevalece of Reibacterium salmoiarum i Chiook salmo (Ocorhychus tshawytscha). Aquaculture 117: Meade, J. W High loadig rates weake immue respose i cultured lake trout. U.S. Fish ad Wildlife Service, Research Iformatio Bulleti 86-70, Wellsboro, Pesylvaia. Nicholso, L. C., L. C. Woods, ad J. G. Woiwode Itesive culture techiques for the striped bass ad its hybrids. Pages i R. M. Harrell, J. H. Kerby, ad R. V. Mito, editors. Culture ad propagatio of striped bass ad its hybrids. America Fisheries Society, Bethesda, Marylad. Pickerig, A. D., ad T. G. Pottiger Crowdig causes prologed leucopeia i salmoid fish, despite iterreal acclimatio. Joural of Fish Biology 30: Plumb, J. A., J. M. Grizzle, H. E. Youg, A. D. Noyes, ad S. Lamprecht A iridovirus isolated from wild largemouth bass. Joural of Aquatic Aimal Health 8: Plumb, J. A., ad D. Zilberg The lethal dose of largemouth bass virus i juveile largemouth bass ad the comparative susceptibility of striped bass. Joural of Aquatic Aimal Health 11: SAS Istitute Base SAS 9.1 procedures guide. SAS Istitute, Cary, North Carolia. Smith, C. E., ad W. G. Williams Experimetal itrite toxicity i raibow trout ad Chiook salmo. Trasactios of the America Fisheries Society 103: Taylor, P. W., ad S. D. Roberts Clove oil: A alterative aaesthetic for aquaculture. North America Joural of Aquaculture 61: Wedemeyer, G. A., The physiology of fish i itesive culture. Chapma ad Hall, New York. Williams, T The iridoviruses. Advaces i Virus Research 46: Wolf, K., Fish viruses ad fish viral diseases. Corell Uiversity Press, Ithaca, New York.